In vitro diagnostic assays have been performed with microspheres for over twenty years. The microspheres include microparticles, beads, polystyrene beads, microbeads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles and colored beads. The microspheres serve as vehicles for molecular reactions. Microspheres for use in flow cytometry are obtained from manufacturers, such as Luminex Corp. of Austin, Tex.
Illustrative microspheres and methods of manufacturing same are, for example, found in U.S. patent application Ser. No. 09/234,841 to Mark B. Chandler and Don J. Chandler, entitled Microparticles with Multiple Fluorescent Signals, and in U.S. patent application Ser. No. 09/172,174 to Don J. Chandler, Van S. Chandler, and Beth Lambert, entitled Precision Fluorescently Dyed Particles and Methods of Making and Using Same, both patent applications incorporated herein by reference in their entirety. By way of example, if a user were performing an Ig G, A, M Isotyping Assay, the user opts for bead sets, such as Luminex 8070 IgG, 8060 IgA, and 8050 IgM bead sets.
Microspheres or beads range in diameter from 10 nanometers to 100 microns and are uniform and highly spherical. Bead-based assays are embodied in a standard "strip test," where beads coated with a capture reactant are fixed to a location on a paper strip and beads with another reactant occupy another position on the same paper strip. When a target analyte is introduced to the strip, the first bead type attaches to it and flows or mixes with the second, often causing a color change which indicates the presence of the target analyte.
More recent bead-based assays use flow cytometry to measure reactions with target analytes of interest. In conventional flow cytometers, as shown in FIG. 1, sample biological fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sample fluid stream is injected into, at, or near, the center of the flow cell or cuvette 1910. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in suspension in the flow cell.
A laser diode 1900 focuses a laser beam on them as they pass through the laser beam by a flow of a stream of the suspension. Laser diodes in conventional flow cytometers often require shaping a round beam into an elliptical beam to be focused on the flow cell 1910. As shown in FIG. 1, this elliptical beam is often formed from the round beam using beam shaping optics 1960 located between the laser diode 1900 and the flow cell 1910.
When an object of interest in the flow stream is struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in FIG. 1, light scatter detectors 1930, 1932 are located opposite the laser diode 1900, relative to the flow cell 1910, to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative size and refractive property of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., incorporated herein by reference, have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., incorporated herein by reference, some flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a laser diode 1900, and then emits light at a longer wavelength. FIG. 1 depicts a prior art flow cytometer which uses beam splitters 1942, 1944, 1946 to direct light from the flow cell 1910 to photo-multiplier and filter sets 1956, 1958, 1959 and to side light scatter detector 1932. This flow cytometer employs a mirror 1970 to reflect forward light scatter to forward light scatter detector 1930.
In a standard flow cytometric competitive inhibition assay, by way of example, an antibody is covalently bound to microspheres. These beads are mixed with a biological sample along with a fluorescenated antigen. In the presence of an antigen of interest, the fluorescenated antigen competes for space on the beads, while in its absence, the fluorescenated antigen envelops the bead. Upon examination by flow cytometry, the presence of the antigen of interest is indicated by a marked decrease in fluorescence emission relative to a sample which contains the antigen of interest.
I have determined that there is, however, a stark contrast between these two types of bead-based assays. The former is simple and inexpensive, but is limited to crude assays with strong sample concentrations of the analyte of interest. The latter is powerful and highly sensitive, but requires a $100,000 instrument and a highly trained technician to run the assay and interpret the results.
I have recognized that there is no commercially available instrument that bridges the gap between these two types of bead-based assays. I have determined that an apparatus that combines the sensitivity and flexibility of flow cytometric assays with the simplicity and low cost of strip assays would advance the art of in vitro diagnostics.
I have recognized that much of the cost and size of a flow cytometer is attributable to the laser. Virtually all commercial flow cytometers use an argon ion 488 nm laser as an excitation source. It is large, occupying several cubic feet, requires a massive power supply, and needs constant forced air cooling to maintain stability. There are other smaller and less expensive lasers, but I have ascertained that they are unsuitable for flow cytometry. For example, dye lasers burn out too quickly. He-Cd lasers are too noisy. Frequency doubled lasers are too weak. The He-Ne laser is reasonably effective, but its red output is not the color of choice in flow cytometry.
In view of the shortcomings of the above-mentioned lasers, I have assessed the merits of laser diodes. However, I have determined that the problem with diode lasers is their beam profiles. FIG. 2a, by way of example, shows a sample beam profile of a standard laser diode. The beam profile of the laser diode is very uneven as compared to that of a standard argon ion laser, as shown, by way of example, in FIG. 2b.
I have recognized that the unevenness presents a significant obstacle for flow analyzers because associated fluorescence measurements depend upon substantially uniform excitation among particles and cells. This obstacle can be explained with reference to FIG. 3, which shows, by way of example, a two-dimensional graph of a major axis of the laser diode beam profile depicted in FIG. 2a. I have determined that if the major axis of the beam profile of FIG. 3 lies across a flow path of a flow analyzer, objects in the flow stream, such as cells or microspheres are not subject to light having the same or substantially the same energy levels. Rather, as shown in FIG. 3, points 10, 12, 14, 16, and 18 on the graph have energy levels that vary indiscriminately across the beam profile.
I have determined that if a microsphere is passing through the flow stream and subject to the laser diode beam at, for example, point 10 on the graph of the beam profile would get much more energy than, if the same microsphere were passing through the flow stream and subject to the laser diode beam at point 14. As such, I have recognized that it is impossible to distinguish between a microsphere having a high fluorescence intensity passing through a point on the beam profile having a low energy level or a microsphere having a low fluorescence intensity passing through a point on the beam profile having a high fluorescence intensity.
Commercial flow cytometers, that offer diode lasers as a second laser to accompany the argon ion laser, take for granted the large coefficients of variation (CVs) of the beam profile of the diode laser. Moreover, laser diodes need not have identical beam profiles. Indeed, even minor differences in resonating cavities, for example, affect the shape of respective beam profiles. Thus, a diode laser in a flow cytometer of a given model need not have the same beam profile of a diode laser in another flow cytometer of the same model.
As such, commercial flow cytometers, as shown by way of example, in FIG. 1, employ beam shaping optics, such as prismatic expanders, beam shaping expanders, and micro lens arrays. Prior art implementations of diode lasers in flow cytometry have attempted to optically correct the beam, steering the two outside peaks toward the center.
I have determined that such optics are unnecessarily expensive by themselves, and add to the manufacturing complexity of the flow cytometers, which, in turn, further adds to the overall cost of the instrument. Moreover, I have determined that despite the expensive and complex beam shaping optics employed, the resulting beam profile is still unsatisfactory, as shown in FIG. 4. Although the beam profile in FIG. 4 is better than that shown in FIG. 2a, for example, it still yields a ten to fifteen percent variation in energy intensity across the flow path.
In view of the above, I have determined that it would be desirable to have a method and/or apparatus for providing precise measurements of light scatter and fluorescence by accommodating an uneven beam profile of a diode laser.
I have also determined that it would be desirable to have such a method and/or system absent beam shaping optics optically cooperating with or coupled to the laser diode.
I have further determined that it would be desirable to have such a method and/or system including a flow analyzer.